The present invention relates to expression of immunoglobulins in plants that contain a protection protein as well as to transgenic plants that express such immunoglobulins. The therapeutic use of these immunoglobulins is also contemplated.
Monoclonal antibodies have great potential for numerous therapeutic purposes. The advantages of monoclonal antibody therapeutics over conventional pharmaceuticals include their exquisite selectivity, multiple effector functions, and ease of molecular manipulation such as radioisotope labelling and other types of conjugation. A wide variety of target antigens have been used to generate specific monoclonal antibodies. See for example Therapeutic Monoclonal Antibodies, C. A. K. Borrebaeck and J. W. Larrick eds., Stockton Press, New York, 1990, and The Pharmacology of Monoclonal Antibodies, M. Rosenberg and G. P. Moore eds., Springer-Verlag, Berlin, 1994.
One therapeutic application of monoclonal antibodies is passive immunotherapy in which the exogenously produced immunoglobulins are administered directly to the animal being treated by injection or by ingestion. To be successful, passive immunotherapy must deliver an appropriate amount of an immunoglobulin to the animal, because passive immunotherapy does not rely on an immune response in the animal being treated. The immunoglobulins administered must be specific for the pathogen or molecule desired to effect treatment. One advantage of passive immunotherapy is the speed at which the antibody can be contacted with the target compared to a normal immune response. Passive immunotherapy can also be used as a prophylaxis to prevent the onset of diseases or infections.
A major potential use of passive immunotherapy is in combating bacterial infections. Recent emergence of anti-biotic resistant bacteria make treatment of bacterial infections with passive immunotherapy desirable. Antibiotic treatment targeted to a single pathogen often involves eradication of a large population of normal microbes, and this can have undesired side effects. An alternative approach has been to utilize the inherent specificity of immunoglobulins to inhibit a specific pathogenic function in very specific microbial populations. In this strategy, purified immunoglobulins of the appropriate specificity would be administered in order to provide a passive barrier to pathogen invasion.
In addition, the immunoglobulins used for passive immunotherapies for example, for oral administration of immunoglobulins must meet certain requirements. First, the immunoglobulin must be functional in very harsh environments, such as the gastrointestinal tract. Second, the immunoglobulin must be resistant to the actions of proteases so that it will not be degraded prior to inactivating the target.
Certain types of cells, including epithelial cells and hepatocytes, are capable of assembling immunoglobulin molecules which have been specifically adapted to function in harsh environments. These immunoglobulins are referred to as secretory immunoglobulins (SIg) and include both secretory IgA (SIgA) and secretory IgM (SIgM). The protection provided by endogenous secretory immunoglobulins have been demonstrated. Several mechanisms for protection from bacterial infection by secretory immunoglobulins have been proposed, including, but not limited to, direct killing, agglutination, inhibition of epithelial attachment and invasion, inactivation of enzymes and toxins, opsonization, and complement activation. In an animal, endogenously produced SIgA are exposed to very harsh environments where numerous proteases, such as intestinal and bacterial enzymes are extremely active and denaturants, such as stomach acid, are also present.
One component of secretory immunoglobulins, the secretory component, helps to protect the immunoglobulin against these inactivating agents thereby increasing the biological effectiveness of secretory immunoglobulin.
The mechanism of synthesis and assembly of these secretory immunoglobulins, such as SIgA or SIgM is extremely complex. In animal cells, secretory immunoglobulins are assembled in a process involving different cells types. Each secretory immunoglobulin is made up of immunoglobulin heavy and light chains, joining chain (J chain) and a secretory component. The immunoglobulin producing B cells make and assemble the immunoglobulin heavy and light chain together with J chain to produce dimeric or polymeric IgM or IgA. The secretory component is produced by a second type of cell, either epithelial cells or hepatocytes, and secretory immunoglobulin is assembled in and secreted from these cells. The mechanism by which these cells assemble and secrete the secretory immunoglobulin is extremely complex and requires a unique microenvironment provided, for example, by mucosal tissues. The microenvironment places the B cells that produce the polymeric immunoglobulin near the cells that assemble and secrete secretory immunoglobulin onto the mucosal surface of an animal.
The epithelial cells have a receptor, the polyimmunoglobulin receptor (pIgR), that specifically recognizes and binds polymeric immunoglobulin/containing J chain, internalizing it and transporting it through the epithelial cell. Expressed on the basolateral cell surface, the pIgR has an N-terminal signal peptide of 18 amino acids, an extracellular polyimmunoglobulin binding portion of 629 amino acids, a membrane spanning segment of 23 hydrophobic residues, and a cytoplasmic tail of 103 amino acids. The extra-cellular portion contains five immunoglobulin-like domains of 100-111 amino acids each and constitutes the secreted form of the molecule. See for example, Mostov, Ann. Rev. Immol., 12:63-84 (1994) The site at which the polyimmunoglobulin receptor is cleaved to generate mature secretory component has not been accurately determined.
The polyimmunoglobulin receptor is located on the basolateral surface of epithelial cells in animals. Polymeric, J chain-containing immunoglobulins produced in B cells interact with and are bound by the receptor resulting in vesicularization, transport across the epithelial cell, and ultimate secretion to the mucosal surface. Transepithelial transport also involves proteolysis and phosphorylation to produce the mature SIg containing the secretory component. The close association of the required cells found in the mucosal microenvironment, specifically the B lymphocytes and epithelial cells, is required for secretory immunoglobulin assembly.
The targeting of the production of immunoglobulins in transgenic organisms, such as mice, is extremely difficult and transgenic organisms made from fungus or plants do not contain the proper cell types and mucosal microenvironment to produce secretory immunoglobulins. The production of large amounts of secretory immunoglobulins in transgenic organisms and cell culture has, before this invention, been impossible. One desiring to produce a secretory immunoglobulin in cell culture or a transgenic organism must express the immunoglobulin heavy chain, the immunoglobulin light chain, and J chain in a B lymphocyte. To mimic the proper mucosal microenvironment a cell having the pIgR receptor on its surface would also have to be present and be in close association with that B lymphocyte to even attempt to assemble a functional secretory immunoglobulin.
This elaborate process required for natural secretory immunoglobulin assembly is extremely difficult to duplicate in cell culture or transgenic organisms. Production of SIg in cell culture or transgenic organisms would require coupling the functions of cells producing immunoglobulin with the functions of epithelial cells in artificial (in vitro) systems. Moreover, if the desired transgenic organism is a fungus, a bacterium, or a plant, the cell types and pathways of receptor-mediated cellular internalization, transcytosis, and secretion simply are not present. Those organisms lack epithelial cells and the required mucosal microenvironment.
To date only the assembly of immunoglobulins having light, heavy and J chain within the same cell has been reported. See Carayannopoulos et al. Proc. Nat Acad. Sci., U.S.A., 91:8348-8352 (1994). However, the assembly of an immunoglobulin having the additional protein component, secretory component, within a single cell has not been described.
The present invention discloses a novel method for the assembly of these complex molecules. Rather than assemble the tetrameric complex at the epithelial cell surface by the interaction of a membrane bound polyimmunoglobulin receptor with immunoglobulin, we have assembled secretory immunoglobulin composed of alpha, J, and kappa immunoglobulin chains associated with a protection protein derived from pIgR. This invention produces transgenic plants that assemble secretory immunoglobulins with great efficiency. The present invention makes passive immunotherapy economically feasible.
The present invention contemplates a new type of immunoglobulin molecule. Immunoglobulins of the present invention contain a protection protein in association with an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain. In other embodiments, the immunoglobulin of the present invention further comprise an immunoglobulin derived light chain having at least a portion of an antigen binding domain associated with the immunoglobulin derived heavy chain.
The protection proteins of the present invention give the immunoglobulins containing these protein useful properties including resistance to chemical and enzymatic degradation and resistance to denaturation. These protection proteins enhanced the resistance of the immunoglobulins to environmental conditions.
The protection proteins of the proteins of the present invention comprise at least a segment of amino acid residues 1 to 606 of native polyimmunoglobulin receptor (pIgR) of any species. Other useful protection proteins include protection proteins that contain portions of the pIgR molecule. For example, the protection protein may comprise all or part of: amino acids 1-118 (domain I of rabbit pIgR), amino acids 1 to 223 (domains I and II of rabbit pIgR); amino acids 1 to 332 (domains I, II, III of rabbit pIgR); amino acids 1 to 441 (domains I, II, III, and IV rabbit of pIgR); amino acids 1 to 552 (domains I, II, III, IV and V of rabbit pIgR); and amino acids 1 to 606 or 1 to 627 of pIgR. Additional amino acids, derived either from the pIgR sequence 653-755, or from other sources, may be included so long as they do not constitute a functional transmembrane spanning segment.
In other preferred embodiments, the immunoglobulins of the present invention have a protection protein which has a first amino acid sequence which substantially corresponds to at least a portion of the amino acid residues 1 to 606 or 1 to 627 of the rabbit polyimmunoglobulin receptor and has a second amino acid residue sequence contiguous with said first amino acid sequence, wherein said second amino acid residue sequence does not have an amino acid residue sequence corresponding to the transmembrane segment of the rabbit polyimmunoglobulin receptor.
In more preferred embodiments, the second amino acid residue sequence has at least a portion of an amino acid sequence which corresponds to amino acid residues 655 to 755 of a polyimmunoglobulin receptor. In other preferred embodiments, the second amino acid residue is at least a portion of one or more of the following: an intracellular domain of a polyimmunoglobulin molecule, a domain of a member of the immunoglobulin gene superfamily, an enzyme, a toxin, or a linker.
The present invention contemplates protection proteins which do not have an amino acid residue corresponding to the transmembrane segment of rabbit polyimmunoglobulin receptor but may have amino acid residues corresponding to the intracellular domain of the rabbit polyimmunoglobulin receptor and this are deletion mutants of the receptor.
The present invention also contemplates immunoglobulins containing protection proteins which have an amino acid sequence which does not contain amino acid residues of a polyimmunoglobulin receptor from a species which are analogous to amino acid residues 288 to 755 of the rabbit immunoglobulin receptor, but does contain at least a portion of the amino acid residues or the domains from a polyimmunoglobulin receptor of a species which are analogous to one or more of these amino acid segments: Amino acids corresponding to amino acid residues 20-45 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues 1 to 120 of the rabbit polyimmunoglobulin receptor: amino acids corresponding to or analogous to amino acid residues numbers 120-230 of the rabbit immunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues numbers 230-340 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues 340-456 of the rabbit polyimmunoglobulin receptor; amino acids corresponding to or analogous to amino acid residues numbers 450-550 to 570 of the rabbit polyimmunoglobulin receptors; amino acids corresponding to or analogous to amino acid residues 550 to 570-606 to 627 of the rabbit polyimmunoglobulin receptor.
The protection proteins of the present invention may be derived from many species and include protection proteins derived from mammals, rodents, humans, bovine, porcine, ovine, fowl, caprine, mouse, rat, guinea pig, chicken or other bird and rabbit.
In preferred embodiments, the immunoglobulins of the present invention contain two or four immunoglobulin derived heavy chains having at least a portion of an antigen binding domain associated with the protection protein and two or four immunoglobulin derived light chains having at least a portion of an antigen binding domain bound to the each of the immunoglobulin derived heavy chains.
In other preferred embodiments, the immunoglobulins of the present invention further comprise immunoglobulin J chain bound to at least one of the immunoglobulin derived heavy chains. In preferred embodiments, the component parts of the immunoglobulins of the present invention are bound together by hydrogen bonds, disulfide bonds, covalent bonds, ionic interactions or combinations of said bonds. In other preferred embodiments, the immunoglobulin of the present invention contain protection proteins and/or immunoglobulin derived heavy, light or J chains that are free from N-linked and/or O-linked oligosaccharides.
The immunoglobulins of the present invention may be used as therapeutic immunoglobulins against, for example, mucosal pathogen antigens. In preferred embodiments, the immunoglobulins of the present invention are capable of preventing dental caries by binding to an antigen from S. mutans serotypes c, e and f; and S. sobrinus stereotype d and g, using older nomenclature S. mutans a, c, d, e, f, g and h.
The present invention also contemplates a eukaryotic cell, including a plant cell, containing an immunoglobulin of the present invention. Eukaryotic cells, including plant cells, containing a nucleotide sequence encoding a protection protein and a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain is also contemplated. Eukaryotic cells, including plant cells, that additionally contain a nucleotide sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain is also contemplated. In preferred embodiments, the eukaryotic cells, including plant cells, of the present invention contain nucleotide sequences that encode immunoglobulins that have an antigen binding domain is capable of binding an antigen from S. mutans serotypes a, c, d, e, f, and g, h (S. mutans serotypes c, e and f and S. sobrinus serotypes d and g under new nomenclature. The nucleotide sequences include RNA and appropriate DNA molecules arranged for expression.
In preferred embodiments, the plant cells of the present invention are part of a plant such as a whole plant. The present invention contemplates the use of all types of plants, both dicotyledonous and monocotyledonous including alfalfa, and tobacco.
The present invention also contemplates compositions comprising an immunoglobulin of the present invention and plant macromolecules derived from one of the plants useful in practicing the present invention. Particularly contemplated are compositions containing ribulose bisphosphate carboxylase, light harvesting complex, pigments, secondary metabolites or chlorophyll and an immunoglobulin of the present invention. Preferred compositions have an immunoglobulin concentration of between 0.001% and 99.9% mass excluding water. In more preferred embodiments, the immunoglobulin concentrations present in the composition is between 0.1% and 99%. Other preferred compositions have plant macromolecules present in a concentration of between 1% and 99% mass excluding water.
The present invention also contemplates methods for making an immunoglobulin of the present invention comprising introducing into a plant cell an expression vector having a nucleotide sequence encoding a protection protein operably linked to a transcriptional promoter; and introducing into the same plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, operably linked to a transcriptional promoter. Other methods that further include the step of introducing into the same plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin derived light chain having at least a portion of an antigen binding domain, operably linked to a transcriptional promoter. Other preferred methods include also introducing into a plant cell an expression vector containing a nucleotide sequence encoding an immunoglobulin J chain operably linked to a transcriptional promoter.
The present invention also contemplates methods for producing assembled immunoglobulins having heavy, light and J chains and a protection protein by introducing into a eukaryotic cell nucleotide sequences operatively linked for expression to encode an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin light chain having at least a portion of an antigen binding domain, and immunoglobulin J chain, and a protection protein. The method further comprises maintaining the eukaryotic cell under conditions allowing the production and assembly of the immunoglobulin derived heavy and light chains together with the immunoglobulin J chain and the protection protein to form an immunoglobulin containing a protection protein.
The present invention also contemplates methods of making an immunoglobulin resistant to various environmental conditions (more stable) and harsh conditions by operatively linking a nucleotide sequence encoding at least a portion of a desirable antigen binding domain derived from an immunoglobulin heavy chain to a nucleotide sequence encoding at least one domain derived from an immunoglobulin xcexc or xcex1 (IgM or IgA) heavy chain (or other immunoglobulin having increased stability in the environment) to form a nucleotide sequence encoding a chimeric immunoglobulin heavy chain and expressing that nucleotide sequence in a eukaryotic which also contains at least one molecule from the following list: a protection protein, an immunoglobulin derived light chain having at least a portion of an antigen binding domain and an immunoglobulin J chain. The method further comprises allowing the chimeric immunoglobulin heavy chain to assemble with the other molecule present in the same cell to form an immunoglobulin which is resistant to environmental conditions and more stable.
The large scale production of immunoglobulins of the present invention is contemplated by growing the plants of the present invention and extracting the immunoglobulins from those plants. In preferred embodiments, the method of producing therapeutic immunoglobulin compositions containing plant macromolecules includes the step of shearing under pressure a portion of a plant of the present invention to produce a pulp containing a therapeutic immunoglobulin and plant macromolecules in an liquid derived from the apoplast or symplast of the plant and solid plant derived material. Further processing steps are contemplated which include separating the solid plant derived material from the liquid and using a portion of the plant including a leaf, stem, root, tuber, flower, fruit, seed or entire plant. The present invention contemplates the use of a mechanical device or enzymatic method which releases liquid from the apoplast or symplast of said plant followed optionally by separating using centrifugation, settling, flocculation or filtration.
The present invention contemplates immunoglobulins that are chimeric and thus they contain immunoglobulin domains derived from different immunoglobulin molecules. Particularly preferred are immunoglobulins containing domains from IgG, IgM and IgA.
The present invention contemplates immunoglobulins where the immunoglobulin derived heavy chain is comprised of immunoglobulin domains from two different isotopes of immunoglobulin. In preferred embodiments, the immunoglobulin domains used include at least the CH1, CH2, or CH3 domain of mouse IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, or IgD or the Cvar domain. In other preferred embodiments, the immunoglobulin heavy chain is comprised of at least the Cxcexc1, Cxcexc2, Cxcexc3 or Cxcexc4 domain of mouse IgM.
The present invention also contemplates immunoglobulin derived heavy chains made up of immunoglobulin domains include at least the CH1, CH2, or CH3 domain of a human IgG, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD; or least the Cxcexc1, Cxcexc2, Cxcexc3 or Cxcexc4 domain of human IgM; or the Cvar domain. The use of immunoglobulin domains derived from mammals, animals or rodents including any IgG isotype, any Iga isotype, IgE, IgM or IgD is contemplated.
The present invention also contemplates tetratransgenic organisms which are comprised of cells containing four different transgenes each encoding a different polypeptide of a multipeptide molecule wherein at least one of those peptides is associated together to form a multipeptide molecule. The transgenic organisms contemplated by the present invention include transgenic organisms which contain as one of the four transgenes present a transgene encoding a protection protein. The protection protein present in the transgenic organism""s cells is able to assemble together with immunoglobulin heavy chains when present to form immunoglobulins which contain the protection protein.
In preferred transgenic organisms, the cells of the organism express four transgenes which encode an immunoglobulin derived heavy chain having at least a portion of an antigen binding domain, an immunoglobulin derived light chain having at least a portion of an antigen binding domain, an immunoglobulin J chain, and a protection protein. In other preferred transgenic organisms, the cells contain a transgene which encodes a chimeric immunoglobulin heavy chain, an immunoglobulin heavy chain derived form an IgA heavy chain, an immunoglobulin derived from an IgM heavy chain or an immunoglobulin derived from some other isotype of heavy chain.
In the most preferred embodiment, the transgenic organisms of the present invention are a plant. Various types and species of plants are contemplated by the present invention. In addition, the present invention also contemplates mammals which are transgenic organisms containing the various molecules of the present invention. Mammalian transgenic organisms are contemplated by the present invention and include mammalian transgenic organisms which contain four transgenes encoding different polypeptides.